Synaptic dysfunction is a key feature of Alzheimer's disease (AD) and closely correlates with memory impairment. Activity-dependent production of Ap, and targeted accumulation of Ap oligomers at the synapse have been reported, however underlying molecular mechanisms remain poorly understood. We have previously suggested that presenilin (PSI) can adopt one of two conformations -termed open and closed, based on the other protein constituents of the gamma-secretase complex (Serneels et al., 2009,), on the effects of GAMMA-secretase modulators (Uemura et al., 2010), or due to familial AD-related mutations in PSI (Berezovska et al., 2005). However, whether these are different gamma-complexes that are in equilibrium with one another within a cell, or PSI can dynamically change its conformation within a specific complex, is unclear. Furthermore, events and/or protein(s) that may be involved in regulation of PSI conformation and function in the cell remain unknown. Now we established a unique conformation-sensitive FRET-based assay in living cells, and our new preliminary data show that conformation of PSI changes rapidly and reversibly in concert with synaptic activity. Aim1 will build on this observation and extend it to PSI-APP interactions at the synapse. To search for potential activity-dependent modulators of PSI conformation, we performed a proteomics screen of mouse brain lysates in the presence or absence of calcium, and identified several novel PSI interactors, including synaptotagminsi and 9 (Sytl .9), and synapsini (Svnl). Since Syt1 and Synl showed strong but opposing Ca-dependent profiles of interaction with PSI, we will direct our initial attention to these as likely candidates to modulate PSI/gamma-secretase and its interaction with APP at the synapse in an activity-gamma-controlled manner. Intriguingly, Sytl and 9 were also identified in an independent proteomics screen performed by Dr. Kovacs as APP-interacting proteins (Project 4). Aim 2 will test the hypothesis that Ca2+ influx serves as a switch between the two PSI conformational states in the synaptosomal compartment by controlling PSI interactions with synaptic proteins. Aim 3 follows on the idea that PSI can undergo rapid and reversible changes in conformation to examine the role of pharmacological interventions in allosteric modulation of PSI/gamma-secretase (collaboration with Projects 1 and 2), and its interactions with synaptic proteins in vitro and in vivo. Understanding this issue is of high importance both because the closed PSI conformation is associated with increased Ap42 (the AD-associated cleavage) and because manipulation of the PS1 interactions with Sytl and Synl may be translated into therapeutics with a focus on the synapse.